Statistical experimental design to assess the influence of enzymes of nematophagous fungi versus helminths Fabio Ribeiro Braga a,b, *, Filippe Elias de Freitas Soares c , Juliana Milani Araujo a , Leandro Abreu da Fonseca b , Emy Hiura b , Maylla Garschagen Gava b , Fernanda Toledo Vieira b , Jeanne Saraiva da Paz b , Lorendane Milena de Carvalho a , João Victor Faccini a , José Humberto de Queiroz c , Jackson Victor Araújo a,1 a Departamento de Veterinária, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil b Universidade Vila Velha, Espírito Santo, Brazil c Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil ARTICLE INFO Article history: Received 17 April 2014 Accepted 8 September 2014 Keywords: Biological control Helminths Plackett–Burman Fungal enzymes A B ST R AC T The present work used Plackett–Burman experimental design to assess the influence of enzymes of nematophagous fungi versus Strongyloides westeri and trichostrongylides larvae and Platynosomum fastosum eggs. The variables studied in the Plackett–Burman design were the proteases and chitinases of AC001 or VC4 as destructive agents of S. westeri and trichostrongylides larvae, and P. fastosum eggs. All tested enzymes had a significant effect (P < 0.05) on the destruction of S. westeri larvae. Furthermore, only VC4 and AC001 proteases showed a significant effect (P < 0.05) on the destruction of trichostrongylides larvae. On the other hand, chitinases of VC4 showed the highest significance (P < 0.05) on the destruction of P. fastosum eggs. It is proposed that statistical planning for the use of enzymes derived from nematophagous fungi is a viable way to elucidate some questions about their mechanism of action. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Nematophagous fungi are natural enemies of gastrointestinal hel- minth parasites and have been studied as an important strategy to be incorporated into an integrated control system to combat the par- asitic helminth forms present in the environment (Braga et al., 2008a, 2008b; Dias et al., 2012; Paz-Silva et al., 2011). Their way of ‘at- tacking’ the infective larvae and/or helminth eggs, and their production of vegetative structures (conidia and/or chlamydo- spores), have long been known (Barron, 1977; Gronvold et al., 1996). In more recent studies, their enzyme production has been inves- tigated (Yang et al., 2011; Van Ooij 2011; Soares et al., 2011, 2013). According to Soares et al. (2013), proteases produced by nematophagous fungi are effective in the infection process of hel- minth larvae, hydrolyzing their cuticle. With regard to helminth eggs, whose main component is chitin, a number of studies have dem- onstrated the role of chitinases in the destruction of these eggs (Lysek, 1976; Lýsek and Ste ˘rba, 1991). On the other hand, there is a lack of studies using statistical design to study the interaction of these enzymes together on different stages of helminths of domes- tic animals. In this context, the Plackett–Burman statistical experimental design (Plackett and Burman, 1946) can be used to verify which enzyme (protease and/or chitinase) has higher significance in the destruction process of eggs and larvae of helminths. This plan pro- vides a quick and effective way to identify important factors among a large number of variables, thus saving time and keeping convinc- ing information about each parameter (Abdel-Fattah et al., 2005). The aim of this study was to use a statistical experimental design to assess the interaction of enzymes of nematophagous fungi versus Strongyloides westeri and trichostrongylides larvae and Platynosomum fastosum eggs. 2. Materials and methods 2.1. Fungi Two species of nematophagous fungi were used, Duddingtonia flagrans (AC001) and Pochonia chlamydosporia (VC4). These iso- lates were kept in test tubes at 4 °C containing 2% corn-meal-agar (2% CMA) in the dark for 10 days. These fungi were derived from the mycology collection of the Laboratory of Parasitology of the Vet- erinary Department of the Federal University of Viçosa, Minas Gerais, Brazil. * Corresponding author. Universidade Vila Velha, Espírito Santo, Brazil. Tel.: +55 31 38991464; fax: +55 31 38991464. E-mail address: fabioribeirobraga@hotmail.com (F.R. Braga). 1 Scholarship CNPq. http://dx.doi.org/10.1016/j.rvsc.2014.09.005 0034-5288/© 2014 Elsevier Ltd. All rights reserved. Research in Veterinary Science ■■ (2014) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Fabio Ribeiro Braga, et al., Statistical experimental design to assess the influence of enzymes of nematophagous fungi versus helminths, Research in Veterinary Science (2014), doi: 10.1016/j.rvsc.2014.09.005 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc